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Catalytic Mechanism of Cyclic Di-GMP-Specific Phosphodiesterase: a Study of the EAL Domain-Containing RocR from Pseudomonas aeruginosa

Division of Chemical Biology and Biotechnology,¹ Division of Computational and Structural Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore²

Received 1 February 2008/ Accepted 4 March 2008

EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO-1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg²⁺ ion. In addition to the five essential residues involved in Mg²⁺ binding, we propose that the essential residue E₃₅₂ functions as a general base catalyst assisting the deprotonation of Mg²⁺-coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in the k_cat or K_m, leading us to propose their roles in residue positioning and substrate binding. With functions assigned to the conserved groups in the active site, we discuss the molecular basis for the lack of activity of some characterized EAL domain proteins and the possibility of predicting the phosphodiesterase activities for the vast number of EAL domains in bacterial genomes in light of the catalytic mechanism.

Published ahead of print on 14 March 2008.